KLF4 is a zinc finger transcription factor and part of a family that includes oncogenes and tumor suppressor genes. Project 3 has shown that KLF4 is upregulated in a majority of breast cancers and shows properties of an oncogene, consistent with recent studies showing its role in the pluripotent stem cell phenotype. We demonstrate that KLF4 functions as an oncogene by regulating the transcription and function of Notchl (Ntc1) pathway proteins. In addition, KLF4 induces expression of retinoid receptors that function similarly to tumor suppressor genes in breast cancer. Our genetic and pharmacologic studies show that these two KLF4-regulated pathways are critical determinants of KLF4 function. Either inhibition of Ntc1 using y- secretase inhibitors or administration of an RXR-selective retinoid efficiently blocked the transforming activity of KLF4 in vitro or in vivo. These effects were KFL4-specific and were not observed for control oncogenes. We have shown that KLF4 and Ntc1 are prognostic factors that are concordantly expressed in human breast tumors. We used antibodies to these two proteins as combination prognostic factors in breast cancer. These studies suggest that tumors with increased KLF4-Ntc1 pathway activity are clinically aggressive, and that KLF4 and Ntc1 antibodies can be use to assess prognosis in breast cancer (Aim 1). Whereas Ntc1 induces transformation through the Classical Pathway (i.e., CSL and MAML1), KLF4 suppresses CSL and shunts Ntc1 to an Alternate Pathway. When expressed in human mammary MCF10A cells, KLF4 or Ntc1 induced a block to acinar maturation in 3D cultures. KLF4 blocked the epithelialization that normally occurs by day 6 following inoculation of MCF10A cells onto a Matrigel layer. For improved preclinical testing of small molecules we will use MCF10A cells to develop a model in which cells are transformed by the Ntc1 Alternate Pathway (Aim 2). Small molecules that inhibit Ntc1 or promote retinoid receptor signaling blocked transformation by KLF4. Using in vitro breast epithelial models, human breast cancer xenografts, and a rapid model of ErbB2-induced breast cancer that we developed, these small molecules will be tested alone and in combination as inhibitors of KLF4 effects in breast cancer (Aim 3). In clinical trials, we will explore the effects of retinoid receptor signaling on the KLF4-Ntc1 pathway and estimate the anti-tumor efficacy of y-secretase inhibitors in metastatic breast cancer (Aim 4). In the efficacy trial, KLF4-Ntc1 pathway components will be correlated with tumor response or resistance to identify which patients are most likely to benefit. Relevance to public health: Our studies will test whether two specific inhibitors of KLF4 can be used alone or in combination for treatment of breast cancer, and whether KLF4-Ntc1 pathway analysis can predict which patients are likely to have recurrence/relapseand which patients may respond to KLF4-Ntc1 inhibitors.